7-azoniabicyclo[2.2.1]heptane derivatives, methods of production, and pharmaceutical uses thereof

ABSTRACT

Muscarinic acetylcholine receptor antagonists and methods of using them for the treatment of muscarinic acetylcholine receptor-mediated diseases, such as pulmonary diseases, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 13/512,873,filed Oct. 16, 2012, which is a National Stage entry of InternationalApplication Serial No. PCT/US11/022760, filed Jan. 27, 2011, whichclaims the benefit of U.S. Provisional Application No. 61/386,450, filedSep. 24, 2010, and U.S. Provisional Application No. 61/336,952, filedJan. 28, 2010, each of which applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to 7-azoniabicyclo[2.2.1]heptane derivatives,pharmaceutical compositions of the derivatives, and the use thereof intreating muscarinic acetylcholine receptor mediated diseases of therespiratory tract.

BACKGROUND OF THE INVENTION

Acetylcholine released from cholinergic neurons in the peripheral andcentral nervous systems affects many different biological processesthrough interaction with two major classes of acetylcholinereceptors—the nicotinic and the muscarinic acetylcholine receptors.Muscarinic acetylcholine receptors (mAChRs) belong to the superfamily ofG-protein coupled receptors that have seven transmembrane domains. Thereare five subtypes of mAChRs, termed M₁-M₅, and each is the product of adistinct gene. Each of these five subtypes displays uniquepharmacological properties. Muscarinic acetylcholine receptors arewidely distributed in vertebrate organs where they mediate many vitalfunctions. Muscarinic receptors can mediate both inhibitory andexcitatory actions. For example, in smooth muscle located in theairways, M₃ mAChRs mediate contractile responses. For a review, seeCaufield, Pharmac. Ther. 58, 319 (1993).

In the lung, mAChRs have been localized to smooth muscle in the tracheaand bronchi, the submucosal glands, and the parasympathetic ganglia.Muscarinic receptor density is greatest in parasympathetic ganglia andthen decreases in density from the submucosal glands to tracheal andthen bronchial smooth muscle. Muscarinic receptors are nearly absentfrom the alveoli. For a review of mAChR expression and function in thelungs, see Fryer and Jacoby, Am. J. Respir. Crit. Care Med. 158, 154(1998).

Three subtypes of mAChRs have been identified as important in the lungs,M₁, M₂ and M₃ mAChRs. The M₃ mAChRs, located on airway smooth muscle,mediate muscle contraction. Stimulation of M₃ mAChRs activates theenzyme phospholipase C via binding of the stimulatory G protein Gq/11(Gs), leading to liberation of phosphatidyl inositol-4,5-bisphosphate,resulting in phosphorylation of contractile proteins. M₃ mAChRs are alsofound on pulmonary submucosal glands. Stimulation of this population ofM₃ mAChRs results in mucus secretion.

M₂ mAChRs make up approximately 50-80% of the cholinergic receptorpopulation on airway smooth muscles. Although the precise function isstill unknown, they inhibit catecholaminergic relaxation of airwaysmooth muscle via inhibition of cAMP generation. Neuronal M₂ mAChRs arelocated on postganglionic parasympathetic nerves. Under normalphysiologic conditions, neuronal M₂ mAChRs provide tight control ofacetylcholine release from parasympathetic nerves Inhibitory M₂ mAChRshave also been demonstrated on sympathetic nerves in the lungs of somespecies. These receptors inhibit release of noradrenaline, thusdecreasing sympathetic input to the lungs.

M₁ mAChRs are found in the pulmonary parasympathetic ganglia where theyfunction to enhance neurotransmission. These receptors have also beenlocalized to the peripheral lung parenchyma, however their function inthe parenchyma is unknown.

Muscarinic acetylcholine receptor dysfunction in the lung has been notedin a variety of different pathophysiological states. In particular, inasthma and chronic obstructive pulmonary disease (COPD), inflammatoryconditions lead to loss of inhibitory M₂ muscarinic acetylcholineautoreceptor function on parasympathetic nerves supplying the pulmonarysmooth muscle, causing increased acetylcholine release following vagalnerve stimulation (Fryer et al., Life Sci. 64, 449 (1999)). This mAChRdysfunction results in airway hyperreactivity and hyperresponsivenessmediated by increased stimulation of M₃ mAChRs. Thus the identificationof potent mAChR antagonists would be useful as therapeutics in thesemAChR-mediated disease states.

COPD is an imprecise term that encompasses a variety of progressivehealth problems including chronic bronchitis and emphysema, and it is amajor cause of mortality and morbidity in the world. Smoking is themajor risk factor for the development of COPD; nearly 50 million peoplein the U.S. alone smoke cigarettes, and an estimated 3,000 people takeup the habit daily. As a result, COPD is expected to rank among the topfive diseases as a world-wide health burden by the year 2020 Inhaledanticholinergic therapy is currently considered the “gold standard” asfirst line therapy for COPD (Pauwels et al., Am. J. Respir. Crit. CareMed. 163, 1256 (2001)).

Despite the large body of evidence supporting the use of anticholinergictherapy for the treatment of airway hyperreactive diseases such as COPD,relatively few anticholinergic compounds are available for use in theclinic for pulmonary indications. More specifically, in the UnitedStates, ipratropium (Atrovent; also as Combivent in combination withalbuterol) and tiotropium (Spiriva) are currently the only inhaledanticholinergics marketed for the treatment of hyperreactive airwaydiseases. While the latter is a potent and long-acting anti-muscarinicagent, it is not available as a combination with other pharmacologicalagents such as albuterol. This appears to be due to the lack ofsufficient chemical stability of tiotropium in the presence of certainadditional agents.

Thus, there remains a need for novel anticholinergic agents, i.e.,agents that inhibit the binding of acetylcholine to its receptors, whichcan be co-formulated with other pharmaceuticals and which can beadministered conveniently, such as once a day, for the treatment ofhyperreactive airway diseases such as asthma and COPD.

Since mAChRs are widely distributed throughout the body, the ability toapply anticholinergic agents locally and/or topically to the respiratorytract is particularly advantageous, as it would allow for lower doses ofthe drug to be utilized. Furthermore, the ability to design topicallyactive drugs that have long duration of action, and in particular, areretained either at the receptor or by the lung, would avoid unwantedside effects that may be seen with systemic anticholinergic exposure.However, other muscarinic acetylcholine receptor-mediated diseasesrespond to systemic administration. Thus, medications useful forrespiratory disorders can be administered systemically when appropriatefor treatment of the respiratory disorder, or when appropriate fortreatment of a non-respiratory disorder.

SUMMARY OF THE INVENTION

This invention provides for compounds useful for treating, and methodsof treating, a muscarinic acetylcholine receptor (mAChR) mediateddisease, which method comprises administering an effective amount of astereochemically pure compound of Formula (I) or Formula (II).

This invention also relates to compounds which inhibit the binding ofacetylcholine to its receptors. This invention also relates to methodsof inhibiting the binding of acetylcholine to its receptors in a subjectin need thereof which comprises administering to aforementioned subjectan effective amount of a stereochemically pure compound of Formula (I)or Formula (II).

The present invention also provides for the novel stereochemically purecompounds of Formula (I) or Formula (II), and pharmaceuticalcompositions comprising a stereochemically pure compound of Formula (I)or Formula (II), and a pharmaceutically acceptable excipient, carrier,or diluent.

In one embodiment, the invention provides compounds having thestructures shown by Formula (I):

where R₁ is phenyl or thienyl, optionally substituted with alkyl,alkoxy, halo or COOR groups;

R₂ is R₁, cyclopentyl, cyclohexyl, 1-alkylcyclopentyl or1-alkylcyclohexyl;

or R₁ and R₂ together can be 9-xanthenyl or 9-hydroxyxanthenyloptionally substituted on either or both benzene rings with alkyl,alkoxy, halo or COOR groups;

or the group R₁R₂R₃C can be 10-phenothiazinyl optionally substituted oneither or both benzene rings with alkyl, alkoxy, halo or COOR groups;

R₃ is H, or OH;

R₄ and R₅ are lower alkyl, alkoxycarbonylalkyl, aralkyl, or aryloxyalkyl(the latter two optionally substituted with alkyl, alkoxy, halo or thegroup COOR) or together form a five- or six-membered ring optionallysubstituted with aryl or aryloxy;

R is lower alkyl; and

X⁻ represents a pharmaceutically acceptable anion associated with thepositive charge of the N atom, including but not limited to chloride,bromide, iodide, sulfate, methanesulfonate, benzenesulfonate, andtoluenesulfonate. X⁻ can be a monovalent or polyvalent anion.

In another embodiment, the invention provides a compound of Formula (I),wherein the compound is stereochemically pure.

In one embodiment, R₁ is independently selected from phenyl, optionallysubstituted with alkyl, alkoxy, halo or COOR groups, such as —C₁-C₈alkyl, —O—C₁-C₈ alkyl, —F, —Cl, —Br, —I, or —C(═O)—O—C₁-C₄ alkyl groups.In another embodiment, R₁ is unsubstituted phenyl.

In one embodiment, R₂ is cyclopentyl.

In one embodiment, R₃ is OH.

In one embodiment, and R₅ are independently selected from C₁-C₄ alkyl.In another embodiment, both R₄ and R₅ are methyl.

In one embodiment, the invention embraces an isolated compound ofFormula (I), optionally additionally comprising a pharmaceuticallyacceptable carrier or excipient, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces an isolated, stereochemically pure compound of Formula (I),optionally additionally comprising a pharmaceutically acceptable carrieror excipient, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents exclude(s)compounds (5), (6), (7), and/or (8) as defined herein, or an alternatesalt thereof. The foregoing embodiments may optionally also add theproviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In one embodiment, the invention provides a stereochemically purecompound of the structure shown by Formula (II):

where A is independently selected from the group consisting of:

—C(R₁)(R₂)(R₃),

where R₁ is independently selected from phenyl or thienyl, optionallysubstituted with alkyl, alkoxy, halo or COOR groups, such as —C₁-C₈alkyl, —O—C₁-C₈ alkyl, —F, —Cl, —Br, —I, or —C(═O)—O—C₁-C₄ alkyl groups;

where R₂ is independently selected from phenyl, thienyl, cyclopentyl,cyclohexyl, 1-hydroxycyclopentyl or 1-hydroxycyclohexyl, where phenyl,thienyl, cyclopentyl, cyclohexyl, 1-hydroxycyclopentyl or1-hydroxycyclohexyl are optionally substituted with alkyl, alkoxy, haloor COOR groups, such as —C₁-C₈ alkyl, —O—C₁-C₈ alkyl, —F, —Cl, —Br, —I,or —C(═O)—O—C₁-C₄ alkyl groups; and

where R₃ is H or OH;

9-xanthenyl or 9-hydroxyxanthenyl, optionally substituted on either orboth benzene rings with alkyl, alkoxy, halo or COOR groups, such as—C₁-C₈ alkyl, —O—C1-C₈ alkyl, —F, —Cl, —Br, —I, or —C(═O)—O—C₁-C₄ alkylgroups; and

10-phenothiazinyl, optionally substituted on either or both benzenerings with alkyl, alkoxy, halo or COOR groups, such as —C₁-C₈ alkyl,—O—C₁-C₈ alkyl, —F, —Cl, —Br, —I, or —C(═O)—O—C₁-C₄ alkyl groups;

R₄ and R₅ are independently selected from lower alkyl (such as C₁-C₄alkyl), alkoxycarbonylalkyl (such as —C₁-C₈ alkyl-O—(C═O)—C₁-C₈ alkyl),aralkyl (such as —C₁-C₈ alkyl-C₆-C₁₀ aryl), or aryloxyalkyl (such as—C₁-C₈ alkyl-O—C₆-C₁₀ aryl), where alkoxycarbonylalkyl and aralkyl canbe optionally substituted with alkyl, alkoxy, halo or the group COOR(such as —C₁-C₈ alkyl, —O—C₁-C₈ alkyl, —F, —Cl, —Br, —I, or—C(═O)—O—C₁-C₄ alkyl groups) or together form a five- or six-memberedring optionally substituted with aryl (such as —C₆-C₁₀ aryl) or aryloxy(such as —O—C₆-C₁₀ aryl);

R is lower alkyl; and

X⁻ represents a pharmaceutically acceptable anion, including but notlimited to chloride, bromide, iodide, sulfate, methanesulfonate,benzenesulfonate, and toluenesulfonate. X⁻ can be a monovalent orpolyvalent anion.

In one embodiment, A is the group —C(R₁)(R₂)(R₃). In another embodiment,R₁ is independently selected from phenyl, optionally substituted withalkyl, alkoxy, halo or COOR groups, such —C₁-C₈ alkyl, —O—C₁-C₈ alkyl,—F, —Cl, —Br, —I, or —C(═O)—O—C₁-C₄ alkyl groups. In another embodiment,R₁ is unsubstituted phenyl.

In one embodiment, A is the group —C(R₁)(R₂)(R₃). In another embodiment,R₂ is cyclopentyl.

In one embodiment, A is the group —C(R₁)(R₂)(R₃). In another embodiment,R₃ is OH.

In one embodiment, R₄ and R₅ are independently selected from C₁-C₄alkyl. In another embodiment, both R₄ and R₅ are methyl.

In one embodiment, the invention embraces an isolated compound ofFormula (II), optionally additionally comprising a pharmaceuticallyacceptable carrier or excipient, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces an isolated, stereochemically pure compound of Formula (II),optionally additionally comprising a pharmaceutically acceptable carrieror excipient, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents exclude(s)compounds (5), (6), (7), and/or (8) as defined herein, or an alternatesalt thereof. The foregoing embodiments may optionally also add theproviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

Included in the scope of this invention is each active, stereochemicallypure isomer of a compound of Formula (I) or Formula (II), includingcrystalline forms, amorphous forms, hydrates, or solvates. The inventionincludes each isolated, stereochemically pure compound of Formula (I) orFormula (II).

The invention also embraces a pharmaceutical formulation comprising astereochemically pure compound of Formula (I) or Formula (II) and apharmaceutically acceptable carrier or excipient, and optionally one ormore other therapeutic agents. The foregoing embodiments may optionallyalso add the proviso that the one or more other therapeutic agentsexclude(s) compounds (5), (6), (7), and/or (8) as defined herein, or analternate salt thereof. The foregoing embodiments may optionally alsoadd the proviso that the one or more other therapeutic agents is notanother compound of Formula (I) and/or Formula (II).

In one embodiment, the pharmaceutically acceptable anion associated withany of the compounds disclosed herein is selected from the groupconsisting of acetate, besylate (benzenesulfonate), benzoate,bicarbonate, bitartrate, bromide, calcium edentate, camphorsulfonate(camsylate), carbonate, chloride, chlorotheophyllinate, citrate,edetate, ethanedisulfonate (edisylate), ethanesulfonate (esylate),fumarate, gluceptate (glucoheptonate), gluconate, glucuronate,glutamate, hexylresorcinate, hydroxynaphthoate, hippurate, iodide,isethionate, lactate, lactobionate, lauryl sulfate (estolate), malate,maleate, mandelate, mesylate, methanesulfonate, methylnitrate,methylsulfate, mucate, naphthoate, napsylate, nitrate, octadecanoate,oleate, oxalate, pamoate, pantothenate, phosphate, polygalacturonate,salicylate, stearate, succinate, sulfate, sulfosalicylate, tannate,tartrate, teoclate, toluenesulfonate (tosylate), and trifluoroacetate.The anion can be a monovalent anion or a polyvalent anion.

The invention also embraces a method of using the present compounds,such as a stereochemically pure compound of Formula (I) or Formula (II),for treating a variety of indications, including but not limited todiseases mediated by muscarinic acetylcholine receptors. The inventionalso embraces a method of using the present compounds, such as astereochemically pure compound of Formula (I) or Formula (II), fortreating respiratory tract disorders such as chronic obstructivepulmonary disorder (COPD, also called chronic obstructive lung disease),chronic bronchitis, asthma, chronic respiratory obstruction, pulmonaryfibrosis, pulmonary emphysema, rhinorrhea, allergic rhinitis,occupational lung diseases including pneumoconiosis (such as black lungdisease, silicosis and asbestosis), acute lung injury (ALI), and acuterespiratory distress syndrome (ARDS). Other, non-respiratory medicalconditions that can be treated with muscarinic receptor antagonistsinclude, but are not limited to, genitourinary tract disorders, such asurinary urge incontinence, overactive bladder or detrusor hyperactivityand their symptoms; gastroesophageal reflux disease (GERD);gastrointestinal tract disorders, such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; and the like.

In another embodiment, the invention embraces a stereochemically purecompound of the formula:

(1S,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane with anionX⁻;

(1R,2S,)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane with anionX⁻;

(1R,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane with anionX⁻; or

(1S,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane with anionX⁻;

where X⁻ is a pharmaceutically acceptable anion. X⁻ can be a monovalentanion or a polyvalent anion. Each compound can optionally additionallycomprise a pharmaceutically acceptable carrier or excipient, andoptionally additionally comprise one or more other therapeutic agents.The foregoing embodiments may optionally also add the proviso that theone or more other therapeutic agents exclude(s) compounds (5), (6), (7),and/or (8) as defined herein, or an alternate salt thereof. Theforegoing embodiments may optionally also add the proviso that the oneor more other therapeutic agents is not another compound of Formula (I)and/or Formula (II).

In another embodiment, the invention embraces a stereochemically purecompound of the formula:

(1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane with anionX⁻;

(1R,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane with anionX⁻;

(1S,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane with anionX⁻; and

(1S,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane with anionX⁻;

where X⁻ is a pharmaceutically acceptable anion. X⁻ can be a monovalentanion or a polyvalent anion. Each compound can optionally additionallycomprise a pharmaceutically acceptable carrier or excipient, andoptionally additionally comprise one or more other therapeutic agents.The foregoing embodiments may optionally also add the proviso that theone or more other therapeutic agents exclude(s) compounds (1), (2), (3),and/or (4) as defined herein, or an alternate salt thereof. Theforegoing embodiments may optionally also add the proviso that the oneor more other therapeutic agents is not another compound of Formula (I)and/or Formula (II).

In another embodiment, the invention embraces specific compounds of theformula:

(1S,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide;

(1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide;

(1R,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide;and

(1S,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide; instereochemically pure form. Each compound can optionally additionallycomprise a pharmaceutically acceptable carrier or excipient, andoptionally additionally comprise one or more other therapeutic agents.The foregoing embodiments may optionally also add the proviso that theone or more other therapeutic agents exclude(s) compounds (5), (6), (7),and/or (8) as defined herein, or an alternate salt thereof. Theforegoing embodiments may optionally also add the proviso that the oneor more other therapeutic agents is not another compound of Formula (I)and/or Formula (II).

In another embodiment, the invention embraces specific compounds of theformula:

(1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide;

(1R,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide;

(1S,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy 2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide; and

(1S,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy 2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide; in stereochemically pure form.Each compound can optionally additionally comprise a pharmaceuticallyacceptable carrier or excipient, and optionally additionally compriseone or more other therapeutic agents. The foregoing embodiments mayoptionally also add the proviso that the one or more other therapeuticagents is not another compound of Formula (I) and/or Formula (II).

In another embodiment, the invention embraces a composition consistingessentially of a compound of the formula:

(1S,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide;

(1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide

(1R,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy 2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide;

(1S,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide; instereochemically pure form, or consisting essentially of a mixture of(1), (2), (3), and (4) in any proportion, such as a 1:1:1:1 proportion.Each compound or mixture can optionally additionally consist essentiallyof one or more other therapeutic agents. The foregoing embodiments mayoptionally also add the proviso that the one or more other therapeuticagents exclude(s) compounds (5), (6), (7), and/or (8) as defined herein,or an alternate salt thereof. The foregoing embodiments may optionallyalso add the proviso that the one or more other therapeutic agents isnot another compound of Formula (I) and/or Formula (II).

In one embodiment, the invention embraces an isolated compound offormula (1), optionally additionally comprising a pharmaceuticallyacceptable excipient or carrier, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces a stereochemically pure compound of formula (1), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. In one embodiment, the invention embraces anisolated, stereochemically pure compound of formula (1), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents exclude(s)compounds (5), (6), (7), and/or (8) as defined herein, or an alternatesalt thereof. The foregoing embodiments may optionally also add theproviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In one embodiment, the invention embraces an isolated compound offormula (2), optionally additionally comprising a pharmaceuticallyacceptable excipient or carrier, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces a stereochemically pure compound of formula (2), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. In one embodiment, the invention embraces anisolated, stereochemically pure compound of formula (2), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents exclude(s)compounds (5), (6), (7), and/or (8) as defined herein, or an alternatesalt thereof. The foregoing embodiments may optionally also add theproviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In one embodiment, the invention embraces an isolated compound offormula (3), optionally additionally comprising a pharmaceuticallyacceptable excipient or carrier, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces a stereochemically pure compound of formula (3), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. In one embodiment, the invention embraces anisolated, stereochemically pure compound of formula (3), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents exclude(s)compounds (5), (6), (7), and/or (8) as defined herein, or an alternatesalt thereof. The foregoing embodiments may optionally also add theproviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In one embodiment, the invention embraces an isolated compound offormula (4), optionally additionally comprising a pharmaceuticallyacceptable excipient or carrier, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces a stereochemically pure compound of formula (4), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. In one embodiment, the invention embraces anisolated, stereochemically pure compound of formula (4), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents exclude(s)compounds (5), (6), (7), and/or (8) as defined herein, or an alternatesalt thereof. The foregoing embodiments may optionally also add theproviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In one embodiment, the invention embraces an isolated compound offormula (5), optionally additionally comprising a pharmaceuticallyacceptable excipient or carrier, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces a stereochemically pure compound of formula (5), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. In one embodiment, the invention embraces anisolated, stereochemically pure compound of formula (5), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In one embodiment, the invention embraces an isolated compound offormula (6), optionally additionally comprising a pharmaceuticallyacceptable excipient or carrier, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces a stereochemically pure compound of formula (6), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. In one embodiment, the invention embraces anisolated, stereochemically pure compound of formula (6), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In one embodiment, the invention embraces an isolated compound offormula (7), optionally additionally comprising a pharmaceuticallyacceptable excipient or carrier, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces a stereochemically pure compound of formula (7), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. In one embodiment, the invention embraces anisolated, stereochemically pure compound of formula (7), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In one embodiment, the invention embraces an isolated compound offormula (8), optionally additionally comprising a pharmaceuticallyacceptable excipient or carrier, and optionally additionally comprisingone or more other therapeutic agents. In one embodiment, the inventionembraces a stereochemically pure compound of formula (8), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. In one embodiment, the invention embraces anisolated, stereochemically pure compound of formula (8), optionallyadditionally comprising a pharmaceutically acceptable excipient orcarrier, and optionally additionally comprising one or more othertherapeutic agents. The foregoing embodiments may optionally also addthe proviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In another embodiment, the invention comprises a method of treating amuscarinic acetylcholine receptor (mAChR)-mediated disease, comprisingadministering a therapeutically effective amount of a stereochemicallypure compound of Formula (I) or Formula (II) to a subject in need ofsuch treatment. In one embodiment of the method, the compoundadministered is (1), that is, the compound is(1S,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (1) is isolated, andoptionally combined with a pharmaceutically acceptable excipient. Infurther embodiments of the method, the compound (1) is stereochemicallypure, and optionally combined with a pharmaceutically acceptableexcipient. In further embodiments of the method, the compound (1) isisolated and stereochemically pure. In further embodiments of themethod, the compound (1) is isolated and stereochemically pure, and iscombined with a pharmaceutically acceptable excipient. In one embodimentof the method, the compound administered is (2), that is, the compoundis (1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (2) is isolated, andoptionally combined with a pharmaceutically acceptable excipient. Infurther embodiments of the method, the compound (2) is stereochemicallypure, and optionally combined with a pharmaceutically acceptableexcipient. In further embodiments of the method, the compound (2) isisolated and stereochemically pure. In further embodiments of themethod, the compound (2) is isolated and stereochemically pure, and iscombined with a pharmaceutically acceptable excipient. In one embodimentof the method, the compound administered is (3), that is, the compoundis (1R,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (3) is isolated, andoptionally combined with a pharmaceutically acceptable excipient. Infurther embodiments of the method, the compound (3) is stereochemicallypure, and optionally combined with a pharmaceutically acceptableexcipient. In further embodiments of the method, the compound (3) isisolated and stereochemically pure. In further embodiments of themethod, the compound (3) is isolated and stereochemically pure, and iscombined with a pharmaceutically acceptable excipient. In one embodimentof the method, the compound administered is (4), that is, the compoundis (1S,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (4) is isolated, andoptionally combined with a pharmaceutically acceptable excipient. Infurther embodiments of the method, the compound (4) is stereochemicallypure, and optionally combined with a pharmaceutically acceptableexcipient. In further embodiments of the method the compound (4) isisolated and stereochemically pure. In further embodiments of themethod, the compound (4) is isolated and stereochemically pure, and iscombined with a pharmaceutically acceptable excipient. In one embodimentof the method, the compound administered is (5), that is, the compoundis (1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (5) is isolated, andoptionally combined with a pharmaceutically acceptable excipient. Infurther embodiments of the method, the compound (5) is stereochemicallypure, and optionally combined with a pharmaceutically acceptableexcipient. In further embodiments of the method, the compound (5) isisolated and stereochemically pure. In further embodiments of themethod, the compound (5) is isolated and stereochemically pure, and iscombined with a pharmaceutically acceptable excipient. In one embodimentof the method, the compound administered is (6), that is, the compoundis (1R,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (6) is isolated, andoptionally combined with a pharmaceutically acceptable excipient. Infurther embodiments of the method, the compound (6) is stereochemicallypure, and optionally combined with a pharmaceutically acceptableexcipient. In further embodiments of the method, the compound (6) isisolated and stereochemically pure. In further embodiments of themethod, the compound (6) is isolated and stereochemically pure, and iscombined with a pharmaceutically acceptable excipient. In one embodimentof the method, the compound administered is (7), that is, the compoundis (1S,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (7) is isolated, andoptionally combined with a pharmaceutically acceptable excipient. Infurther embodiments of the method, the compound (7) is stereochemicallypure, and optionally combined with a pharmaceutically acceptableexcipient. In further embodiments of the method, the compound (7) isisolated and stereochemically pure. In further embodiments of themethod, the compound (7) is isolated and stereochemically pure, and iscombined with a pharmaceutically acceptable excipient. In one embodimentof the method, the compound administered is (8), that is, the compoundis (1S,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (8) is isolated, andoptionally combined with a pharmaceutically acceptable excipient. Infurther embodiments of the method, the compound (8) is stereochemicallypure, and optionally combined with a pharmaceutically acceptableexcipient. In further embodiments of the method, the compound (8) isisolated and stereochemically pure. In further embodiments of themethod, the compound (8) is isolated and stereochemically pure, and iscombined with a pharmaceutically acceptable excipient. In any of theabove embodiments, the composition may optionally additionally compriseone or more other therapeutic agents; such embodiments may optionallyalso add the proviso that the one or more other therapeutic agents isnot another compound of Formula (I) and/or Formula (II).

In another embodiment, the invention comprises a method of suppressing amuscarinic acetylcholine receptor (mAChR)-mediated disease, byadministering an amount of one or more compounds of Formula (I) orFormula (II) sufficient to partially or totally suppress the disease toa subject in need of such treatment. In one embodiment of the method,the compound administered is (1), that is, the compound is(1S,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (1) is isolated andstereochemically pure. In further embodiments of the method, thecompound (1) is isolated and stereochemically pure, and is combined witha pharmaceutically acceptable excipient. In one embodiment of themethod, the compound administered is (2), that is, the compound is(1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (2) is isolated andstereochemically pure. In further embodiments of the method, thecompound (2) is isolated and stereochemically pure, and is combined witha pharmaceutically acceptable excipient. In one embodiment of themethod, the compound administered is (3), that is, the compound is(1R,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (3) is isolated andstereochemically pure. In further embodiments of the method, thecompound (3) is isolated and stereochemically pure, and is combined witha pharmaceutically acceptable excipient. In one embodiment of themethod, the compound administered is (4), that is, the compound is(1S,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method the compound (4) is isolated andstereochemically pure. In further embodiments of the method, thecompound (4) is isolated and stereochemically pure, and is combined witha pharmaceutically acceptable excipient. In one embodiment of themethod, the compound administered is (5), that is, the compound is(1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (5) is isolated andstereochemically pure. In further embodiments of the method, thecompound (5) is isolated and stereochemically pure, and is combined witha pharmaceutically acceptable excipient. In one embodiment of themethod, the compound administered is (6), that is, the compound is(1R,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (6) is isolated andstereochemically pure. In further embodiments of the method, thecompound (6) is isolated and stereochemically pure, and is combined witha pharmaceutically acceptable excipient. In one embodiment of themethod, the compound administered is (7), that is, the compound is(1S,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (7) is isolated andstereochemically pure. In further embodiments of the method, thecompound (7) is isolated and stereochemically pure, and is combined witha pharmaceutically acceptable excipient. In one embodiment of themethod, the compound administered is (8), that is, the compound is(1S,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide. Infurther embodiments of the method, the compound (8) is isolated andstereochemically pure. In further embodiments of the method, thecompound (8) is isolated and stereochemically pure, and is combined witha pharmaceutically acceptable excipient. In any of the aboveembodiments, the composition may optionally additionally comprise one ormore other therapeutic agents; such embodiments may optionally also addthe proviso that the one or more other therapeutic agents is not anothercompound of Formula (I) and/or Formula (II).

In any of the above methods, the muscarinic acetylcholine receptor(mAChR)-mediated disease can be selected from the group consisting ofrespiratory tract disorders such as chronic obstructive pulmonarydisorder (COPD, also called chronic obstructive lung disease), chronicbronchitis, asthma, chronic respiratory obstruction, pulmonary fibrosis,pulmonary emphysema, rhinorrhea, allergic rhinitis, occupational lungdiseases including pneumoconiosis (such as black lung disease, silicosisand asbestosis), acute lung injury (ALI), acute respiratory distresssyndrome (ARDS), genitourinary tract disorders, such as urinary urgeincontinence, overactive bladder or detrusor hyperactivity and theirsymptoms; gastroesophageal reflux disease (GERD); gastrointestinal tractdisorders, such as irritable bowel syndrome, diverticular disease,achalasia, gastrointestinal hypermotility disorders and diarrhea; andthe like.

In another embodiment, the invention embraces a composition consistingessentially of a mixture of (1) and (4) in any proportion, such as a 1:1proportion. In another embodiment, the invention embraces a compositionconsisting essentially of a mixture of (2) and (3) in any proportion,such as a 1:1 proportion. In another embodiment, the invention embracesa composition consisting essentially of a mixture of (1) and (2) in anyproportion, such as a 1:1 proportion. In another embodiment, theinvention embraces a composition consisting essentially of a mixture of(3) and (4) in any proportion, such as a 1:1 proportion. Any of theforegoing compositions can additionally consist essentially of anoptional additional therapeutic agent. The foregoing embodiments mayoptionally also add the proviso that the one or more other therapeuticagents exclude(s) compounds (5), (6), (7), and/or (8) as defined herein,or an alternate salt thereof. The foregoing embodiments may optionallyalso add the proviso that the one or more other therapeutic agents isnot another compound of Formula (I) and/or Formula (II).

In another embodiment, the invention embraces a composition consistingessentially of a mixture of (1) and (3) in any proportion, such as a 1:1proportion. In another embodiment, the invention embraces a compositionconsisting essentially of a mixture of (2) and (4) in any proportion,such as a 1:1 proportion. Any of the foregoing compositions canadditionally consist essentially of an optional additional therapeuticagent. The foregoing embodiments may optionally also add the provisothat the one or more other therapeutic agents exclude(s) compounds (5),(6), (7), and/or (8) as defined herein, or an alternate salt thereof.The foregoing embodiments may optionally also add the proviso that theone or more other therapeutic agents is not another compound of Formula(I) and/or Formula (II).

In another embodiment, the invention comprises a compound of the formula

exo-2-((R)-2′-cyclopentyl-2′-hydroxy-2′-phenylacetoxy)spiro[bicyclo-[2.2.1]heptane-7,1′-pyrrolidin]-1′-iumanion, where the anion X⁻ is a pharmaceutically acceptable anion.

In another embodiment, the invention comprises a compound of the formula

(exo-2-((R)-2′-cyclopentyl-2′-hydroxy-2′-phenylacetoxy)spiro[bicyclo-[2.2.1]heptane-7,1′-pyrrolidin]-1′-iumbromide (9)).

In another embodiment, the invention comprises a composition comprisingthe compound (9) and a pharmaceutically acceptable excipient or carrier,and optionally additionally comprising one or more other therapeuticagents. In one embodiment, the composition comprising the compound (9)and a pharmaceutically acceptable excipient or carrier also comprisesone or more compounds selected from (1), (2), (3), (4), (5), (6), (7),or (8).

The invention also embraces a method of using the compound (9), eitheralone or in combination with other agents, and optionally comprising apharmaceutically acceptable excipient or carrier, for treating a varietyof indications, including but not limited to diseases mediated bymuscarinic acetylcholine receptors. The invention also embraces a methodof using the compound (9), either alone or in combination with otheragents, and optionally comprising a pharmaceutically acceptableexcipient or carrier, for treating respiratory tract disorders such aschronic obstructive pulmonary disorder (COPD, also called chronicobstructive lung disease), chronic bronchitis, asthma, chronicrespiratory obstruction, pulmonary fibrosis, pulmonary emphysema,rhinorrhea, allergic rhinitis, occupational lung diseases includingpneumoconiosis (such as black lung disease, silicosis and asbestosis),acute lung injury (ALI), and acute respiratory distress syndrome (ARDS).Other, non-respiratory medical conditions that can be treated withmuscarinic receptor antagonists include, but are not limited to,genitourinary tract disorders, such as urinary urge incontinence,overactive bladder or detrusor hyperactivity and their symptoms;gastroesophageal reflux disease (GERD); gastrointestinal tractdisorders, such as irritable bowel syndrome, diverticular disease,achalasia, gastrointestinal hypermotility disorders and diarrhea; andthe like.

Some embodiments described herein are recited as “comprising” or“comprises” various elements. In alternative embodiments, those elementscan be recited with the transitional phrase “consisting essentially of”or “consists essentially of” as applied to those elements. In furtheralternative embodiments, those elements can be recited with thetransitional phrase “consisting of” or “consists of” as applied to thoseelements. Thus, for example, if a composition or method is disclosedherein as comprising A and B, the alternative embodiment for thatcomposition or method of “consisting essentially of A and B” and thealternative embodiment for that composition or method of “consisting ofA and B” are also considered to have been disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the percentage of bronchoprotection in rats provided bycertain compounds of the invention.

FIG. 2 depicts the percentage of bronchoprotection in guinea pigsprovided by certain compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compounds and methods for treating a muscarinicacetylcholine receptor-mediated disease, such as chronic obstructivepulmonary disease (COPD).

DEFINITIONS

By “subject,” “individual,” or “patient” is meant an individualorganism, preferably a mammal, most preferably a human.

“Treating” a disease with the compounds and methods discussed herein isdefined as administering one or more of the compounds discussed herein,with or without additional therapeutic agents, in order to reduce oreliminate either the disease or one or more symptoms of the disease, orto retard the progression of the disease or of one or more symptoms ofthe disease, or to reduce the severity of the disease or of one or moresymptoms of the disease. “Suppression” of a disease with the compoundsand methods discussed herein is defined as administering one or more ofthe compounds discussed herein, with or without additional therapeuticagents, in order to suppress the clinical manifestation of the disease,or to suppress the manifestation of adverse symptoms of the disease. Thedistinction between treatment and suppression is that treatment occursafter adverse symptoms of the disease are manifest in a subject, whilesuppression occurs before adverse symptoms of the disease are manifestin a subject. Suppression may be partial, substantially total, or total.The compounds and methods of the invention can be administered toasymptomatic patients at risk of developing the clinical symptoms of thedisease, in order to suppress the appearance of any adverse symptoms.

“Therapeutic use” of the compounds discussed herein is defined as usingone or more of the compounds discussed herein to treat or suppress adisease, as defined above. A “therapeutically effective amount” of acompound is an amount of the compound, which, when administered to asubject, is sufficient to reduce or eliminate either a disease or one ormore symptoms of a disease, or to retard the progression of a disease orof one or more symptoms of a disease, or to reduce the severity of adisease or of one or more symptoms of a disease, or to suppress theclinical manifestation of a disease, or to suppress the manifestation ofadverse symptoms of a disease. A therapeutically effective amount can begiven in one or more administrations.

“Alkyl” is intended to embrace a saturated linear, branched, cyclic, ora combination of linear and/or branched and/or cyclic hydrocarbon chainand/or ring of carbon atoms. In one embodiment, alkyl groups havebetween 1 and 12 carbon atoms, that is, C₁-C₁₂ alkyl. In anotherembodiment, alkyl groups have between 1 and 8 carbon atoms, that is,C₁-C₈ alkyl. The point of attachment of the alkyl group to the remainderof the molecule can be at any chemically feasible location on thefragment.

“Alkoxy” refers to the group —O-alkyl, for example, —O—C₁-C₁₂ alkyl or—O—C₁-C₈ alkyl.

“Lower alkyl” is synonymous with “C₁-C₄ alkyl,” and is intended toembrace methyl (Me), ethyl (Et), propyl (Pr), n-propyl (nPr), isopropyl(iPr), butyl (Bu), n-butyl (nBu), isobutyl (iBu), sec-butyl (sBu),t-butyl (tBu), cyclopropyl (cyclPr), cyclobutyl (cyclBu),cyclopropyl-methyl (cyclPr-Me) and methyl-cyclopropane (Me-cyclPr),where the C₁-C₄ alkyl groups can be attached via any valence on theC₁-C₄ alkyl groups to the remainder of the molecule.

“Halo” refers to F, Cl, Brand I.

“Aryl” refers to an aromatic hydrocarbon, such as C₆-C₁₀ aromatichydrocarbons including, but not limited to, phenyl and naphthyl.

“Aryloxy” refers to the group —O-aryl.

“Aralkyl” refers to the group-alkyl-aryl.

“Aryloxyalkyl” refers to the group-alkyl-O-aryl.

“Alkoxycarbonylalkyl” refers to the group-alkyl-(C═O)—O-alkyl.

By “isolated” is meant a compound that has been purified in the chemicalsense of reducing unwanted components. Isolation can be about 80% pureor at least about 80% pure, about 90% pure or at least about 90% pure,about 95% pure or at least about 95% pure, about 98% pure or at leastabout 98% pure, about 99% pure or at least about 99% pure, about 99.5%pure or at least about 99.5% pure, or about 99.9% pure or at least about99.9% pure. Isolation percentages are preferably weight percent, but canalso be mole percent. Components that are desired, such aspharmaceutically acceptable excipients, pharmaceutical carriers, oradditional therapeutic agents, are not included when calculating thepercentage of purity of isolation.

By “stereochemically pure compound” is meant a preparation of a compoundwhich contains primarily one stereoisomer out of two or more possiblestereoisomers. A stereochemically pure compound has about 80% or atleast about 80% of a single stereoisomer, about 90% or at least about90% of a single stereoisomer, about 95% or at least about 95% of asingle stereoisomer, about 98% or at least about 98% of a singlestereoisomer, about 99% or at least about 99% of a single stereoisomer,about 99.5% or at least about 99.5% of a single stereoisomer, or about99.9% or at least about 99.9% of a single stereoisomer. Stereochemicalpurity percentages are preferably mole percent, but can also be weightpercent. Reference to a particular stereoisomer of a compound asstereochemically pure, or to a composition comprising, consistingessentially of, or consisting of a stereochemically pure compound, meansthat the preparation of the compound has about 80% or at least about 80%of the referenced stereoisomer, about 90% or at least about 90% of thereferenced stereoisomer, about 95% or at least about 95% of thereferenced stereoisomer, about 98% or at least about 98% of thereferenced stereoisomer, about 99% or at least about 99% of thereferenced stereoisomer, about 99.5% or at least about 99.5% of thereferenced stereoisomer, or about 99.9% or at least about 99.9% of thereferenced stereoisomer.

As an example, the percent isolation of L-alanine, the desiredcomponent, in a mixture containing 25 mg of beta-alanine, 25 mg ofD-alanine, and 50 mg of L-alanine, where beta-alanine and D-alanine areundesired components, would be 50%. The percent stereochemical purity ofL-alanine in that same mixture would be 66.7%, calculated with respectto the total of all stereoisomers of 2-amino propanoic acid (i.e.,alanine; beta-alanine is 3-amino propanoic acid and is not astereoisomer of alanine) (All three molecules have the same molecularweight, and percent by weight and mole percent both yield the samepercentages in this example.) Addition of, for example, 1 gram ofpharmaceutically acceptable carrier and 50 mg of Vitamin C (where thepharmaceutically acceptable carrier and Vitamin Care desired additionalcomponents of the composition) would not affect the percent isolation orpercent stereochemical purity calculated for L-alanine.

The terms “active M₃ muscarinic acetylcholine receptor antagonist” and“active M₃ mAChR antagonist” are synonymous and are used to designate acompound having an IC₅₀ of less than 5 nanomolar or less than about 5nanomolar, preferably less than 3 nanomolar or less than about 3nanomolar, more preferably less than 1 nanomolar or less than about 1nanomolar, still more preferably less than 0.5 nanomolar or less thanabout 0.5 nanomolar, and yet still more preferably less than 0.3nanomolar or less than about 0.3 nanomolar, as measured by theMuscarinic Receptor Radioligand Binding Assay described below in Example2.

“An alternate salt thereof,” when referring to a compound, indicatesthat the counterion of the compound may be replaced with anothercounterion. For example, possible alternate salts of compound (5),(1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide,include the corresponding chloride:(1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane chloride;the corresponding tosylate: (1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptanetoluenesulfonate; etc.

“Consisting essentially of” as used herein is intended as a limitationto the specified materials or steps recited, and also allows inclusionof any unrecited materials or steps that do not materially affect thebasic characteristics of the composition or method. Thus, a compositionconsisting essentially of compound (1) would exclude any other mAChRantagonist compound, such as compounds (2)-(8), from being present inthe mixture, but one or more pharmaceutically acceptable excipients orcarriers suitable for the intended route of administration (e.g., apharmaceutically acceptable excipient or carrier for administration viainhalation, a pharmaceutically acceptable excipient or carrier foradministration via injection, or a pharmaceutically acceptable excipientor carrier for administration via oral administration) would not beexcluded from a composition consisting essentially of compound (1), evenif such a pharmaceutically acceptable excipient or carrier is notexplicitly recited.

It should be appreciated that the structures depicted in Formula (I) andFormula (II) represent at least four possible stereoisomersincorporating the four possible isomers of the7-azabicyclo[2.2.1]heptan-2-ol moieties as illustrated.

If additional stereo centers are present, for example, if for the group—C(R₁)(R₂)(R₃), the carbon atom substituted by R₁, R₂, and R₃ isasymmetric, a total of at least eight different diastereomers willresult.

If the R₄ and R₅ groups are different, additional stereoisomers may begenerated in the quaternization step.

Included in the scope of this invention are all active isomers, mixturesof active isomers, crystalline forms, amorphous forms, hydrates, orsolvates of the subject compounds.

The chemical structures drawn herein and the chemical names listedherein are to be construed as including all isotopologues. Isotopologuesare molecular entities that differ only in isotopic composition (numberof isotopic substitutions), e.g. CH₄, CH₃D, CH₂D₂, etc., where “D” isdeuterium, that is, 2H. Isotopologues can have isotopic replacements atany or at all atoms in a structure, or can have atoms present in naturalabundance at any or all locations in a structure.

Various embodiments of the invention described herein are recited as“comprising” or “comprises” various elements. In alternativeembodiments, those elements can be recited with the transitional phrase“consisting essentially of” or “consists essentially of” as applied tothose elements. In further alternative embodiments, those elements canbe recited with the transitional phrase “consisting of” or “consists of”as applied to those elements. Thus, for example, if a composition ormethod is disclosed herein as comprising A and B, the alternativeembodiment for that composition or method of “consisting essentially ofA and B” and the alternative embodiment for that composition or methodof “consisting of A and B” are also considered to have been disclosedherein.

Methods of Preparation

The compounds of Formula (I) and Formula (II) may be obtained byapplying the appropriate synthetic procedures, some of which areillustrated in the scheme below which is for illustrative purposes only.

As outlined in Scheme 1, the desired compounds of Formula (I) andcertain compounds of Formula (II) can be prepared by transesterificationof ester 1 with the appropriate N-Boc protected amino alcohol 2. Acidtreatment of the resulting ester 3 provides the secondary amine 4 whichis converted to the tertiary amine 5 by N-alkylation utilizing reductiveamination procedures. Finally, quaternization of the tertiary nitrogenof amino ester 5 with an alkyl or aralkyl bromide furnishes thecompounds of Formula (I).

Esters 1 where R₁ and R₂ are phenyl or 2-thienyl and R₃ is OH arecommercially available. Esters 1 where R₁ is phenyl or thienyl, R₂ iscycloalkyl and R₃ is OH can be prepared by reaction of an arylglyoxylate (i.e. PhCOCOOMe) with a cycloalkyl Grignard reagent. Esterswhere R₃ is not OH have also been prepared. The diastereoselectiveaddition of such organometallics to chiral arylglyoxylates leading tooptically active products has been described (Tetrahedron Letters 29,2175 (1988)).

Transesterification of methyl esters 1 with Boc-protected amino alcohols2 to form the amino esters 3 is carried out using a catalytic amount ofa strong base such as sodium hydride, sodium methoxide and the like in asuitable inert solvent such as n-heptane or toluene at temperatures highenough to allow separation of the methanol formed by distillation.

In the case where the group A or R₁R₂R₃C is 10-phenothiazinyl, thedesired ester 3 is prepared by reacting 10-chlorocarbonylphenothiazinewith amino alcohols 2 in the presence of a base.

Removal of the Boc protection group is achieved by treatment with acidto give the secondary amines 4.

The R₄ group, for example methyl, is introduced by reductive aminationwith formaldehyde and sodium triacetoxyborohydride as described in J.Org. Chern. 61, 3489-3862 (1996) to furnish the tertiary amines 5.

Finally, treatment of the tertiary amines with a compound of formula R₅Xin an inert solvent provides the quaternary ammonium compounds ofFormula (I).

Spiroazonia compounds where and R₅ together form a 5- or 6-membered ringcan be prepared from secondary amines 4 by treatment with a dihaloalkanesuch as 1,4-dichlorobutane or 1,5-dibromopentane in the presence of abase such as triethylamine or potassium carbonate in a suitable solvent.

The methodology described in U.S. Pat. No. 4,353,922 and J. Pharm. Sci.74, 208-210 (1985) does not provide the compounds of this invention, asthese methods provide mixtures of compounds rather than isolatedcompounds. For example, it was originally thought this methodologyproduced a mixture of the compounds (3), (4), (6), and (8) describedherein. However, more recent research has conclusively demonstrated thatthe crucial intramolecular epoxide opening step proceeds via anunprecedented cis (not trans) mechanism, leading to the exo aminoalcohol intermediates; see J. Org. Chem. 59, 1771-1778 (1994) and Org.Lett. 1, 1439-1441 (1999). Thus, the endo stereochemistry of compound 4reported in J. Pharm. Sci. 74, 208-210 (1985) (RS-11635, a mixture offour distinct diastereomers rather than a stereochemically purecompound) is incorrect, and compound 4 of J. Pharm. Sci. 74, 208-210(1985) is in fact a mixture of the compounds (1), (2), (5), and (7)described herein.

The procedures listed in the Examples section demonstrate for the firsttime how to synthesize all eight possible individual diastereomers ofFormula (I) where the carbon atom of the group R₁R₂R₃C is an asymmetriccarbon.

Diseases Amenable to Treatment with Compounds of the Invention

The compounds and methods disclosed herein can be used to treat variousdiseases, particularly diseases mediated by muscarinic acetylcholinereceptors. These diseases include, but are not limited to, respiratorytract disorders such as chronic obstructive pulmonary disorder (COPD,also called chronic obstructive lung disease), chronic bronchitis,asthma, chronic respiratory obstruction, pulmonary fibrosis, pulmonaryemphysema, rhinorrhea, allergic rhinitis. The diseases also include, butare not limited to, occupational lung diseases including pneumoconiosis(such as black lung disease, silicosis and asbestosis), acute lunginjury (ALI), and acute respiratory distress syndrome (ARDS).

Additional, non-respiratory medical conditions that can be treated withmuscarinic receptor antagonists include, but are not limited to,genitourinary tract disorders, such as urinary urge incontinence,overactive bladder or detrusor hyperactivity and their symptoms;gastroesophageal reflux disease (GERD); gastrointestinal tractdisorders, such as irritable bowel syndrome, diverticular disease,achalasia, gastrointestinal hypermotility disorders and diarrhea; andthe like.

Methods of Use

The compounds, pharmaceutical compositions, and methods of the inventioncan be used in treatment and/or suppression of muscarinic acetylcholinereceptor mediated diseases, and in one particular embodiment, intreatment and/or suppression of muscarinic acetylcholine receptormediated diseases of the respiratory tract. In particular, thecompounds, pharmaceutical compositions, and methods of the invention canbe used in treating and/or suppressing chronic obstructive pulmonarydisorder (COPD), chronic bronchitis, and/or emphysema.

For treatment of respiratory disorders, the compounds and pharmaceuticalcompositions are preferably administered in a form that can be inhaled,such as in aerosols, mists, sprays, or powders. In one embodiment, thecompounds or pharmaceutical compositions are delivered in a formsuitable for inhalation. The form suitable for inhalation can beparticles, aerosols, powders, or droplets with an average size of about10 μm diameter or less, preferably in the range between about 0.1 μm toabout 5 μm, or in the range between about 1 μm to about 5 μm. See, forexample, Hickey, Anthony J., ed., Pharmaceutical Inhalation AerosolTechnology, 2nd Ed., New York: Marcel Dekker, 2004, particularly PartTwo on Methods of Generation, Administration, and Characterization ofAerosols. Typically, about two-thirds, preferably 80%, more preferably90% of the particles, aerosols, powders or droplets will fall within thesize range specified. For example, when droplets having a diameter inthe range of about 1 μm to about 5 μm are specified, two-thirds,preferably about 80%, more preferably about 90% of the droplets willhave a diameter falling in the range of about 1 μm to about 5 μm.

The compounds or pharmaceutical compositions can be delivered in a formsuitable for inhalation by using various types of inhalers, such as anebulizing inhaler, metered-dose inhalers, or a dry powder inhaler(DPI). The compounds or pharmaceutical compositions can be delivered byvoluntary inhalation by a subject or patient, or by mechanicalventilation if a subject or patient requires assistance in breathing.

The compounds described herein can be formulated as pharmaceuticalcompositions by formulation with additives such as pharmaceuticallyacceptable excipients, pharmaceutically acceptable carriers, andpharmaceutically acceptable vehicles. Suitable pharmaceuticallyacceptable excipients, carriers and vehicles include processing agentsand drug delivery modifiers and enhancers, such as, for example, calciumphosphate, magnesium stearate, talc, monosaccharides, disaccharides,starch, gelatin, cellulose, methyl cellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-β-cyclodextrin,polyvinylpyrrolidinone, low melting waxes, ion exchange resins, and thelike, as well as combinations of any two or more thereof. Other suitablepharmaceutically acceptable excipients are described in “Remington'sPharmaceutical Sciences,” Mack Pub. Co., New Jersey (1991), and“Remington: The Science and Practice of Pharmacy,” Lippincott Williams &Wilkins, Philadelphia, 20th edition (2003) and 21st edition (2005),incorporated herein by reference.

A pharmaceutical composition can comprise a unit dose formulation, wherethe unit dose is a dose sufficient to have a therapeutic or suppressiveeffect. The unit dose may be sufficient as a single dose to have atherapeutic or suppressive effect. Alternatively, the unit dose may be adose administered periodically in a course of treatment or suppressionof a disorder.

Pharmaceutical compositions containing the compounds of the inventionmay be in any from suitable for the intended method of administration,including, for example, a solution, a suspension, or an emulsion. Liquidcarriers are typically used in preparing solutions, suspensions, andemulsions. Liquid carriers contemplated for use in the practice of thepresent invention include, for example, water, saline, pharmaceuticallyacceptable organic solvent(s), pharmaceutically acceptable oils or fats,and the like, as well as mixtures of two or more thereof. The liquidcarrier may contain other suitable pharmaceutically acceptable additivessuch as solubilizers, emulsifiers, nutrients, buffers, preservatives,suspending agents, thickening agents, viscosity regulators, stabilizers,and the like. Suitable organic solvents include, for example, monohydricalcohols, such as ethanol, and polyhydric alcohols, such as glycols.Suitable oils include, for example, soybean oil, coconut oil, olive oil,safflower oil, cottonseed oil, and the like. For parenteraladministration, the carrier can also be an oily ester such as ethyloleate, isopropyl myristate, and the like. Compositions of the presentinvention may also be in the form of microparticles, microcapsules,liposomal encapsulates, and the like, as well as combinations of any twoor more thereof.

Time-release or controlled release delivery systems may be used, such asa diffusion controlled matrix system or an erodible system, as describedfor example in: Lee, “Diffusion-Controlled Matrix Systems”, pp. 155-198and Ron and Langer, “Erodible Systems”, pp. 199-224, in “Treatise onControlled Drug Delivery”, A. Kydonieus Ed., Marcel Dekker, Inc., NewYork 1992. The matrix may be, for example, a biodegradable material thatcan degrade spontaneously in situ and in vivo for, example, byhydrolysis or enzymatic cleavage, e.g., by proteases. The deliverysystem may be, for example, a naturally occurring or synthetic polymeror copolymer, for example in the form of a hydrogel. Exemplary polymerswith cleavable linkages include polyesters, polyorthoesters,polyanhydrides, polysaccharides, poly(phosphoesters), polyamides,polyurethanes, poly(imidocarbonates) and poly(phosphazenes).

The compounds of the invention may be administered enterally, orally,parenterally, sublingually, by inhalation (e.g. as mists or sprays),rectally, or topically in dosage unit formulations containingconventional nontoxic pharmaceutically acceptable carriers, adjuvants,and vehicles as desired. For example, suitable modes of administrationinclude oral, subcutaneous, transdermal, transmucosal, iontophoretic,intravenous, intraarterial, intramuscular, intraperitoneal, intranasal(e.g. via nasal mucosa), intraocular, subdural, vaginal, rectal,gastrointestinal, and the like, and directly to a specific or affectedorgan or tissue, such as the lung or bladder. Topical administration mayalso involve the use of transdermal administration such as transdermalpatches or iontophoresis devices. The term parenteral as used hereinincludes subcutaneous injections, intravenous injection, intramuscularinjection, intrasternal injection, or infusion techniques. The compoundsare mixed with pharmaceutically acceptable carriers, adjuvants, andvehicles appropriate for the desired route of administration. Thecompounds described for use herein can be administered in solid form, inliquid form, in aerosol form, or in the form of tablets, pills, powdermixtures, capsules, granules, injectables, creams, solutions,suppositories, enemas, colonic irrigations, emulsions, dispersions, foodpremixes, and in other suitable forms. The compounds can also beadministered in liposome formulations. The compounds can also beadministered as prodrugs, where the prodrug undergoes transformation inthe treated subject to a form which is therapeutically effective.Additional methods of administration are known in the art.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in propylene glycol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms may also compriseadditional substances other than inert diluents, e.g., lubricatingagents such as magnesium stearate. In the case of capsules, tablets, andpills, the dosage forms may also comprise buffering agents. Tablets andpills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining ineli diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, cyclodextrins, and sweetening,flavoring, and perfuming agents.

The compounds of the present invention can also be administered in theform of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multilamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andphosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.W., p.33 et seq (1976).

In certain embodiments of the invention, the formulations andpreparations of the invention, and the formulations and preparationsused in the methods of the invention, are sterile. Sterilepharmaceutical formulations are compounded or manufactured according topharmaceutical-grade sterilization standards (United States PharmacopeiaChapters 797, 1072, and 1211; California Business & Professions Code4127.7; 16 California Code of Regulations 1751, 21 Code of FederalRegulations 211) known to those of skill in the art.

The invention also provides articles of manufacture and kits containingmaterials useful for treating or suppressing muscarinic acetylcholinereceptor-mediated diseases. The invention also provides kits comprisingany one or more of the compounds of the invention. In some embodiments,the kit of the invention comprises the container described above.

In other aspects, the kits may be used for any of the methods describedherein, including, for example, to treat an individual with a muscarinicreceptor-mediated disease, or to suppress a muscarinic acetylcholinereceptor-mediated disease in an individual.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost to which the active ingredient is administered and the particularmode of administration. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, body area, body mass index (BMI),general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination, and the type,progression, and severity of the particular disease undergoing therapy.The pharmaceutical unit dosage chosen is usually fabricated andadministered to provide a defined final concentration of drug in theblood, tissues, organs, or other targeted region of the body, or toprovide a defined dosage of the drug to a specific site, such as thelungs. The therapeutically effective amount or effective amount for agiven situation can be readily determined by routine experimentation andis within the skill and judgment of the ordinary clinician.

Examples of dosages of the compounds described herein which can be usedare an effective amount within the dosage range of about 0.1 μg to about10 mg per kilogram of body weight, about 0.1 μg to about 5 mg perkilogram of body weight, about 0.1 μg to about 1 mg per kilogram of bodyweight, about 0.1 μg to about 0.5 mg per kilogram of body weight, about0.1 μg to about 100 μg per kilogram of body weight, about 0.1 μg toabout 50 μg per kilogram of body weight, about 0.1 μg to about 10 μg perkilogram of body weight, or about 1 μg to about 10 μg per kilogram ofbody weight. When administered orally or by inhalation, examples ofdosages are an effective amount within the dosage range of about 0.001mg to about 0.01 mg, or about 0.01 mg to about 0.1 mg, or about 0.1 mgto about 1 mg, or about 1 mg to about 10 mg, or about 10 mg to about 100mg, or about 100 mg to about 1 g. Preferred fixed doses include about0.005 mg, about 0.01 mg, about 0.018 mg, about 0.02 mg, about 0.03 mg,about 0.04 mg, about 0.05 mg, about 0.1 mg, about 1 mg, about 2 mg,about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 50 mg, about 80mg or about 100 mg, independently of body weight. However, it isunderstand that pediatric patients may require smaller dosages, anddepending on the severity of the disease and condition of the patient,dosages may vary. The compound will preferably be administered oncedaily, but may be administered two, three or four times daily, or everyother day, or once or twice per week.

Compounds of the present invention may be administered in a single dailydose, or the total daily dosage may be administered in divided dosage oftwo, three or four times daily.

When formulated as a liquid, the concentration of the compound describedherein will typically be about 0.01 mg/ml to about 0.1 mg/ml or about0.1 mg/ml to about 1 mg/ml, but can also be about 1 mg/ml to about 10mg/ml or about 10 mg/ml to about 100 mg/ml. When formulated as a solid,for example as a tablet or as a powder for inhalation, theconcentration, expressed as weight compound divided by total weight,will typically be about 0.01% to about 0.1%, about 0.1% to about 1%,about 1% to about 10%, or about 10% to about 100%.

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more other agents used in the treatment or suppression ofmuscarinic acetylcholine receptor-mediated diseases. Examples ofadditional agents that can be used in combination with the compounds ofthe current invention include, but are not limited to, otheracetylcholine receptor inhibitors, such as ipratropium and tiotropium;or one or more anti-inflammatory, bronchodilator, antihistamine,decongestant or antitussive agents. The additional agents can beadministered simultaneously in the same pharmaceutical composition,simultaneously in different pharmaceutical compositions, or at differenttimes. Specific agents include, but are not limited to, corticosteroidssuch as fluticasone propionate, budesonide, beclomethasone dipropionate,flunisolide, triamcinolone acetonide, ciclesonide, or mometasonefuroate; f32-adrenoreceptor agonists such as albuterol, salmeterol, andmetaproterenol; antitussive agents (cough suppressants) such as codeineor dextromorphan; and theophylline. Desired combinations can bedetermined based on additional therapeutic advantages, potential sideeffects, and other considerations known to the skilled artisan. Someagents can be combined with the compounds of the invention foradministration via inhalation, while others can be administered viaother routes of administration.

When additional active agents are used in combination with the compoundsof the present invention, the additional active agents may generally beemployed in therapeutic amounts as indicated in the Physicians' DeskReference (PDR) 53^(rd) Edition (1999), which is incorporated herein byreference, or such therapeutically useful amounts as would be known toone of ordinary skill in the art.

The compounds of the invention and the other therapeutically activeagents can be administered at the recommended maximum clinical dosage orat lower doses. Dosage levels of the active compounds in thecompositions of the invention may be varied so as to obtain a desiredtherapeutic response depending on the route of administration, severityof the disease and the response of the patient. When administered incombination with other therapeutic agents, the therapeutic agents can beformulated as separate compositions that are given at the same time ordifferent times, or the therapeutic agents can be given as a singlecomposition.

Compounds of Formula (I) and Formula (II), and compounds (1), (2), (3),(4), (5), (6), (7), or (8), by virtue of the quaternary nitrogen, arepositively charged, and thus will have an associated negativecounterion. Any pharmaceutically acceptable anion can be used with thecompounds of the invention, such as those described in Berge et al., J.Phmm. Sci. 66:1 (1977); Bighley et al., “Salt Forms of Drugs andAbsorption,” in Swarbrick J, Boylan J C, eds. Encyclopedia ofPharmaceutical Technology 13, New York, N.Y.: Marcel Dekker;1996:453-499; and Paulekuhn et al., J. Med. Chem. 50:6665 (2007). Theanion can be monovalent (i.e., a charge of −1) or polyvalent (e.g., acharge of −2, −3, etc.). Pharmaceutically acceptable anions include, butare not limited to, acetate, besylate (benzenesulfonate), benzoate,besylate, bicarbonate, bitartrate, bromide, calcium edentate,camphorsulfonate (camsylate), cm.bonate, chlmide, chlorotheophyllinate,citrate, edetate, ethanedisulfonate (edisylate), ethanesulfonate(esylate), fumarate, gluceptate (glucoheptonate), gluconate,glucuronate, glutamate, hexylresorcinate, hydroxynaphthoate, hippurate,iodide, isethionate, lactate, lactobionate, lauryl sulfate (estolate),malate, maleate, mandelate, mesylate, methanesulfonate, methylnitrate,methylsulfate, mucate, naphthoate, napsylate, nitrate, octadecanoate,oleate, oxalate, pamoate, pantothenate, phosphate, polygalacturonate,salicylate, stearate, succinate, sulfate, sulfosalicylate, tannate,tmirate, teoclate, toluenesulfonate (tosylate), and trifluoroacetate.Multiple anions can be used in a single preparation if desired; forexample, one micromole of compound (1) can be combined with one-halfmicromole of chloride ion and one-half micromole of bromide ion.

The compounds of Formula (I) and Formula (II), and compounds (1), (2),(3), (4), (5), (6), (7), or (8), have a single formal positive charge,i.e., they are monovalent cations. The stoichiometry of the anion to thesingly-charged (monovalent) cation will depend on the valency of theanion; e.g., when the anion is a monovalent anion, such as Br—, thecation:anion ratio will be 1:1; when the anion is a divalent anion, suchas sulfate (Sol-), the cation:anion ratio will be 2:1, and so forth.

EXAMPLES Example 1 Synthetic Methods Example 1.1(±)-exo-7-[(tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-ol and(±)-endo-7-[(tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-ol

Palladium on carbon (10%, 1.55 g) and ammonium formate (2.48 g, 39.3mmol) were added to a stirred solution of the combined alcohols(±)-exo-7-(phenylmethyl)-7-azabicyclo[2.2.1]heptan-2-ol and(±)-endo-7-(phenylmethyl)-7-azabicyclo[2.2.1]heptan-2-ol (1.55 g, 7.63mmol) in dry methanol (51 mL). The resulting suspension was stirred atreflux temperature for 20-30 min. After completion, the catalyst wasremoved by filtration through a pad of Celite, which was then washedseveral times with methanol. The filtrate was concentrated in vacuo andto the residue was added anhydrous tetrahydrofuran (17 mL) followed bydi-tert-butyl dicarbonate (2.0 g, 9.16 mmol). The reaction mixture wasstirred at room temperature for 3 hrs and the solvent was concentratedin vacuo. The residue was taken up with methylene chloride and washedwith a solution of ammonium hydroxide. The organic phase was dried(MgSO₄) and concentrated. The residue was purified by flash columnchromatography to give the alcohols(±)-exo-7-[(tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-ol and(±)-endo-7-[(tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-ol (1.50g, 95%).

Example 1.2 (±)-7-[tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-one

Dess-Martin periodinane (3.83 g, 9.03 mmol) was added in severalportions under nitrogen to a stirred solution of the alcohols(±)-exo-7-[(tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-ol and(±)-endo-7-[(tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-ol (1.50g, 7.04 mmol) in anhydrous methylene chloride (125 mL). The reactionmixture was stirred overnight at room temperature. After removal of thesolvent in vacuo, the solid residue was triturated with diethyl etherand filtrated. The solid was washed several times with diethyl ether andthe filtrate was concentrated in vacuo. The residue was purified byflash column chromatography to give the ketone(±)-7-[tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-one (1.06 g,71%).

Example 1.3 (1S,4′R,5′R)-tert-butyl4′5′-diphenyl-7-azaspiro[bicyclo[2.2.1]heptane-2,2′-imidazolidine]-7-carboxylateand (1R,4′R,5′R)-tert-butyl4′,5′-diphenyl-7-azaspiro[bicyclo[2.2.1]heptane-2,2′-imidazolidine]-7-carboxylate

(R,R)-Diphenylethylenediamine (1.14 g, 5.37 mmol) was added undernitrogen to a solution of ketone(±)-7-[tert-butoxycarbonyl]-7-azabicyclo[2.2.1]heptan-2-one (1.06 g,5.02 mmol) in dry methylene chloride (17 mL) containing 4 Å molecularsieves. The reaction mixture was stirred at room temperature for 24 h.Triethylamine (2.8 mL) was added and the molecular sieves were theneliminated by filtration. The filtrate was concentrated in vacuo and theresulting residue purified by flash column chromatography(ether/petroleum ether/Et₃N, 10:15:1 to 15:10:1) affording first(1S,4′R,5′R)-tert-butyl4′,5′-diphenyl-7-azaspiro[bicyclo[2.2.1]heptane-2,2′-imidazolidine]-7-carboxylate(992 mg, 49%) and then (1R,4′R,5′R)-tert-butyl4′,5′-diphenyl-7-azaspiro-[bicyclo[2.2.1]heptane-2,2′-imidazolidine]-7-carboxylate(0.938 g, 46%).

Example 1.4 (+)(1S)-7-(tert-Butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-one

A solution of (1S,4′R,5′R)-tert-butyl4′,5′-diphenyl-7-azaspiro[bicyclo[2.2.1]heptane-2,2′-imidazolidine]-7-carboxylate(992 mg, 2.45 mmol) in 0.1 M H₃PO₄-THF (2:1, 14.4 mL) was stirred for 30min at room temperature. The reaction mixture was then diluted withwater and extracted with ether. The combined extracts were dried (MgSO₄)and the solvent was concentrated in vacuo. The resulting residue waspurified by flash column chromatography to give the ketone (+)(1S)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-one (496 mg,96%); [α]²² _(D)+74.2° (c 0.43, CHCl₃); ¹H NMR δ (CDCl₃, 400 MHz) 4.56(t, 1H), 4.25 (d, 1H), 2.47 (dd, 1H), 1.99 (m+d, 2+1H), 1.59 (m, 2H),1.46 (s, 9H).

Example 1.5(−)-(1R)-7-(tert-Butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-one

Following the procedure described for the preparation of (+)(1S)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-one, thediamine (1R,4′R,5′R)-tert-butyl4′,5′-diphenyl-7-azaspiro[bicyclo[2.2.1]heptane-2,2′-imidazolidine]-7-carboxylate(938 mg, 2.31 mmol) was converted to (−)(1R)-7-(tert-Butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-one (468 mg,96%). [α]²² _(D)-58.6° (c 0.11, CHCl₃); ¹H NMR δ (CDCl₃, 400 MHz) 4.56(t, 1H), 4.25 (d, 1H), 2.47 (dd, 1H), 1.99 (m+d, 2+1H), 1.59 (m, 2H),1.46 (s, 9H).

Example 1.6(1S,2S)-7-(tert-Butoxycarbonyl)-7-azabicyclo[2.2.1]heptane-2-ol and(1S,2R)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol

Platinum oxide (27 mg) followed by triethylamine (0.98 mL, 7.05 mmol)were added to a stirred solution of ketone (+)(1S)-7-(tert-Butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-one (496 mg,2.35 mmol) in ethanol (1.2 mL). The flask was purged under vacuum andwas then filled with hydrogen using a balloon. The reaction mixture wasstirred at room temperature for 48 hours. The catalyst was then removedby filtration through a pad of Celite, which was washed several timeswith methanol. The filtrate was concentrated in vacuo and the resultingresidue was purified by flash column chromatography affording first(1S,2S)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol (140 mg,28%) and then(1S,2R)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol (160 mg,32%).

Example 1.7(1R,2R)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol and(1R,2S)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol

Following the procedure described for the preparation of(1S,2S)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol and(1S,2R)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol, theketone (−) (1R)-7-(tert-Butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-one(468 mg, 2.22 mmol) was converted to(1R,2R)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol (150 mg,32%) and (1R,2S)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptan-2-ol(140 mg, 30%).

Example 1.8 (1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane

To (1R,2S)-2-hydroxy-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane(68 mg, 0.32 mmol) in 5 mL of heptane was added(R)-methyl-2-cyclopentyl-2-hydroxy 2-phenylacetate (149 mg, 0.64 mmol)followed by cat. NaH (8 mg as a 60% dispersion in oil) and the mixturewas stirred at 100° C. for 20 hrs. Water was added and the mixture wasextracted with ethyl acetate, dried over sodium sulfate andconcentrated. The residue was purified by column chromatography onsilica gel using hexane/ethyl acetate as eluent to yield 76 mg of thedesired product, (1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane asan oil.

Similarly prepared were:

-   (1S,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane,-   (1S,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane,-   (1R,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane,-   (1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane,-   (1S,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane,-   (1S,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy 2′-phenyl    acetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane, and-   (1R,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane.

Example 1.9 (1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane

To (1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane (76mg, 0.182 mmol) was added 1 mL of 4N hydrochloric acid in dioxane andthe mixture was stirred at room temperature for 0.5 hr. The dioxane wasremoved under vacuum and the residue was basified with ammoniumhydroxide to pH 10 and extracted with 3×30 mL of methylene chloride. Thecombined organic layers were dried over sodium sulfate and concentratedto yield the crude amine. To this amine, dissolved in 5 mL ofdichloroethane, was added 0.1 mL of formaldehyde solution (37% w/v inwater) followed by sodium triacetoxyborohydride (76 mg, 0.364 mmol) andthe mixture stirred at room temperature overnight. Water was added andthe mixture was extracted with 3×50 mL of methylene chloride. Thecombined organic layers were dried over sodium sulfate and concentratedto yield the crude tertiary amine which was further purified on silicagel using methylene chloride/methanol/ammonia (90:9:1) as the eluent toyield 46 mg of (1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7-methyl-7-azabicyclo[2,2,1]heptane as an oil.

Similarly prepared were:

-   (1S,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane,-   (1S,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane,-   (1R,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane,-   (1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane,-   (1S,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane-   (1S,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane, and-   (1R,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy    2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane.

Example 1.10 (1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide (2)

To (1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7-methyl-7-azabicyclo[2.2.1]heptane (46 mg, 0.139mmol) in acetone (2 mL) was added 1 mL of methyl bromide solution (2M inether). The resulting mixture was left at room temperature for 48 hrs.The crystallized product was filtered off and dried to yield 36 mg of(1R,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide (2)as a white crystalline solid, M.P. 233-234° C., ¹HNMR: 7.6 (dd, 2H); 7.4(m, 2H); 7.28 (m, 1H); 5.0 (m, 1H); 4.9 (m, 1H); 4.45 (m, 1H); 3.8 (s,1H); 3.49 (s, 3H); 3.28 (s, 3H); 3.0 (m, 1H); 2.5-2.2 (m, 4H); 1.9-1.3(m, 10H).

Similarly prepared were:

(1), (1S,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide,M.P. 198-200° C., ¹HNMR: 7.45 (dd, 2H); 7.25 (m, 2H); 7.1 (m, 1H); 5.1(m, 1H); 4.9 (m, 1H); 4.1 (m, 1H); 3.8 (s, 1H); 3.23 (s, 3H); 3.0 (s,3H); 2.8 (m, 1H); 2.3 (m, 4H); 2.8-1.2 (m, 10H);

(4), (1S,2S)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide,M.P. 210-212° C., ¹HNMR: 7.59 (d, 2H); 7.36 (t, 2H); 7.3 (m, 1H); 5.4(m, 1H); 4.8 (m, 1H); 5.6 (m, 1H); 3.6 (s, 1H); 3.5 (s, 6H); 2.85 (m,2H); 2.2 (m, 2H); 1.8-1.2 (m, 9H);

(3), (1R,2R)-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide,M.P. 232-233° C., ¹HNMR: 7.59 (d, 2H); 7.37 (t, 2H); 7.32 (m, 1H); 5.4(m, 1H); 4.9 (t, 1H); 4.4 (t, 1H); 3.6 (s, 1H); 3.56 (s, 3H); 3.46 (s,3H); 3.0 (m, 2H); 2.3 (m, 1H); 1.9 (m, 1H); 1.8-1.2 (m, 10H);

(5), (1R,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide,M.P. 222-224° C., ¹HNMR: 7.6 (dd, 2H); 7.4 (m, 2H); 7.28 (m, 1H); 5.0(m, 1H); 4.9 (m, 1H); 4.45 (m, 1H); 3.8 (s, 1H); 3.49 (s, 3H); 3.28 (s,3H); 3.0 (m, 1H); 2.5-2.2 (m, 4H); 1.9-1.3 (m, 10H);

(7), (1S,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide,M.P. 231-233° C., ¹HNMR: 7.45 (dd, 2H); 7.25 (m, 2H); 7.1 (m, 1H); 5.1(m, 1H); 4.9 (m, 1H); 4.1 (m, 1H); 3.8 (s, 1H); 3.23 (s, 3H); 3.0 (s,3H); 2.8 (m, 1H); 2.3 (m, 4H); 2.8-1.2 (m, 10H);

(8), (1S,2S)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide,M.P. 223-225° C., ¹HNMR: 7.59 (d, 2H); 7.36 (t, 2H); 7.3 (m, 1H); 5.4(m, 1H); 4.8 (m, 1H); 5.6 (m, 1H); 3.6 (s, 1H); 3.5 (s, 6H); 2.85 (m,2H); 2.2 (m, 2H); 1.8-1.2 (m, 9H); and

(6), (1R,2R)-2-((S)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7,7-dimethyl-7-azoniabicyclo[2.2.1]heptane bromide,M.P. 212-214° C., ¹HNMR: 7.59 (d, 2H); 7.37 (t, 2H); 7.32 (m, 1H); 5.4(m, 1H); 4.9 (t, 1H); 4.4 (t, 1H); 3.6 (s, 1H); 3.56 (s, 3H); 3.46 (s,3H); 3.0 (m, 2H); 2.3 (m, 1H); 1.9 (m, 1H); 1.8-1.2 (m, 10H).

Example 1.11Exo-2-((R)-2′-cyclopentyl-2′-hydroxy-2′-phenylacetoxy)spiro[bicyclo-[2.2.1]heptane-7,1′-pyrrolidin]-1′-iumbromide (9)

To a solution of Exo-2-((R)-2′-cyclopentyl-2′-hydroxy2′-phenylacetoxy)-7-azabicyclo[2.2.1]heptane (151 mg, 0.479 mmol) inacetonitrile (3 mL) were added 1,4-dibromobutane (206 mg, 0.958 mmol)and 1,8-diazabicyclo[5.4.0]undec-7-ene (109 mg, 0.717 mmol). Theresulting solution was stirred at 60° C. for 20 hrs. The cooled solutionwas evaporated to dryness and the residual oil was triturated withacetone/ethyl acetate. The resulting solid was recrystallized fromacetone/ethyl acetate, filtered off and dried to give 44 mg of thedesired product, M.P. 214-217° C., MS 370 (M⁺).

Example 2 Biological Methods

The antagonist effects of compounds at the M₃ mAChR of the presentinvention are determined by the following in vitro and in vivo assays.

Muscarinic Receptor Radioligand Binding Assay:

Radioligand binding studies were carried out with M₃ receptor cellhomogenates as described (Peralta et al., The EMBO Journal 6, 3923-3929,(1987)). Incubations of test ligands (or standard) with 0.2 nM[³H]4-DAMP were incubated for 120 minutes at 22° C. using human M₃receptor-expressing cell homogenates. Specific ligand binding to thereceptors was defined as the difference between the total radioligandbinding and the nonspecific binding determined in the presence of anexcess of unlabeled ligand (10 μM atropine). The results were expressedas a percent of control specific binding ((measured specificbinding/control specific binding)×100) obtained in the presence ofvarious concentrations of the test compounds.

The IC₅₀ values (concentration causing a half-maximal inhibition ofcontrol specific binding) and Hill coefficients (nH) were determined bynon-linear regression analysis of the competition curves generated withmean replicate values using Hill equation curve fitting(Y=D+[(A−D)/(1+(C/C₅₀)^(nH))], where Y=specific binding, D=minimumspecific binding, A=maximum specific binding, C=compound concentration,C₅₀=IC₅₀, and nH=slope factor).

The inhibition constants (K_(i)) were calculated using the Cheng-Prusoffequation (K_(i)=IC₅₀/(1+(L/K_(D))), where L=concentration of radioligandin the assay, and K_(D)=affinity of the radioligand for the receptor). AScatchard plot was used to determine the radioligand K_(d).

When tested by the above method, the compounds of the invention hadK_(i) values in the range of 0.1 to 100 nM, as shown in Table 1.

TABLE 1 M₃ (antagonist) % Inhibition Test of Control Compound conc.Specific IC₅₀ K_(i) ID Structure (nM) Binding (nM) (nM) (1)

10 100   0.23  0.16 (2)

10 98  0.25  0.18 (3)

10 99  0.25  0.18 (4)

10 100  0.3  0.22 (5)

10 80 2.1 1.5 (6)

10 75 2.9 2.1 (7)

10 30 19   14   (8)

10  9 36   26  Bronchodilator Potency and Duration of Action Studies; Rat EinthovenModel:

The bronchodilator potency and duration of action studies utilize maleSprague-Dawley rats (200-350 g). Animals are placed in a dosing chamberand exposed to the aerosol generated from an LC Star Nebulizer Set anddriven by a mixture of gases (5% CO₂, 20% oxygen and 75% nitrogen) bybeing placed for no more than 30 min in a dosing chamber. Within thedosing chamber the animals are not restrained but are confined to aspace that has an approximate floor area of 18 square inches. Theanimals are acclimated to the chamber for 10 min, then treated with testcompounds which are delivered via inhalation. Each test compoundsolution is nebulized over 5 to 25 minutes. After a predeterminedperiod, based on the time point studied, the animals are evaluated forthe pharmacodynamic effects of the test compounds. Thirty minutes priorto the start of pulmonary evaluation, the animals are anesthetized withpentobarbital sodium (Nembutal, 25 mg/kg). The jugular vein iscatheterized with saline+10 U/ml heparin-filled polyethylene catheters(PE-20) used to infuse the bronchoconstrictor methacholine (MCh). Thecarotid artery is cannulated with 10 U/ml heparin/saline-filled PE-50catheters and connected to a pressure transducer for the measurement ofblood pressure and heart rate (CV effects). The trachea is thendissected free and cannulated with a 14 G steel tube connected to apressure transducer for the measurement of pulmonary resistance and to aconstant volume rodent respirator set to deliver an appropriate tidalvolume and at a rate determined by the animal's weight. This is used forrat ventilation during the evaluation of the pulmonary and CV effects ofthe test articles. Intravenous MCh is administered at a dose sufficientto cause 80% of the maximal pulmonary constriction in an untreatedanimal (determined by experimentation in a pilot study using 4 rats).The pulmonary and CV responses to the MCh determine the potency, safetyand pharmacodynamic effects of test articles.

Rat Bronchoprotection Protocol-MCh Dose Response:

Test compounds and control (water) were administered to male SpragueDawley rats (200-350 g) via inhalation. Inhalation dosing was done byplacing the rats in a dosing chamber and exposing them for 25 min tonebulized drug solutions using a Pari nebulizer. The animals were thenreturned to their cages. The chamber was decontaminated between uses bywashing with water.

Twenty-four hours after dosing and thirty minutes prior to the start ofpulmonary evaluation, the animals were anesthetized with pentobarbitalsodium (Nembutal, 50 mg/mL, 1 mL/kg, IP). The trachea was then dissectedfree and cannulated with a 14 G steel tube connected to a pressuretransducer (for the measurement of pulmonary inflation pressure) and toa constant volume rodent respirator set to deliver an appropriate tidalvolume (2.5 ml) and at a rate determined by the animal's weight (60breath/min). The carotid artery was cannulated with a 5 U/mlheparin/saline-filled PE-50 cannula and connected to a pressuretransducer for the measurement of blood pressure and heart rate. Thejugular vein was catheterized with a saline filled polyethylene catheter(PE-10) and used to deliver bolus challenges of the bronchoconstrictormethacholine (MCh). Intravenous ascending doses of MCh (1 to 300 μg/kg)were administered, after the response to the previous dose returned tobaseline. The pulmonary inflation pressure and blood pressure wererecorded using a Biopac system with the AcqKnowledge software. Theanimals were euthanized upon completion of the study by cervicaldislocation followed by a thoracotomy.

The results of the bronchoprotection studies are shown in FIG. 1. Table2 shows the in vivo potency (24 h post inhalation) and duration ofbronchoprotective effects against methacholine-inducedbronchoconstriction in rat.

TABLE 2 Compound ID Potency (ID₅₀) Duration (1) ≧10 μg/mL ++ (2) ≦3μg/mL +++ (3) ≦3 μg/mL +++ (4) >10 μg/mL + (5), (6), (7) & (8) >100μg/mL − − inactive 24 h post inhalation + <24 h duration ++ ≧24 hduration +++ ≧48 h durationBronchodilator Potency and Duration of Action Studies; Guinea PigEinthoven Model:

The bronchodilator potency and duration of action studies utilize maleDunkin Hartley guinea pigs (250-350 g). Animals are placed in a dosingchamber and exposed to the aerosol generated from an LC Star NebulizerSet and driven by a mixture of gases (5% CO₂, 20% oxygen and 75%nitrogen) by being placed for no more than 30 min in a dosing chamber.Within the dosing chamber the animals are not restrained but areconfined to a space that has an approximate floor area of 18 squareinches. The animals are acclimated to the chamber for 10 min, thentreated with test compounds which are delivered via inhalation. Eachtest compound solution is nebulized over 5 to 25 minutes. After apredetermined period, based on the time point studied, the animals areevaluated for the pharmacodynamic effects of the test compounds. Thirtyminutes prior to the start of pulmonary evaluation, the animals areanesthetized with intramuscular ketamine (55.8 mg/kg), xylazine (3.9mg/kg) and acepromazine (1 mg/kg). The jugular vein is catheterized withsaline+10 U/ml heparin-filled polyethylene catheters (PE-20) used toinfuse the bronchoconstrictor methacholine (MCh). The carotid artery iscannulated with 10 U/ml heparin/saline-filled PE-50 catheters andconnected to a pressure transducer for the measurement of blood pressureand heart rate (CV effects). The trachea is then dissected free andcannulated with a 14 G steel tube connected to a pressure transducer forthe measurement of pulmonary resistance and to a constant volume rodentrespirator set to deliver an appropriate tidal volume and at a ratedetermined by the animal's weight. This is used for guinea pigventilation during the evaluation of the pulmonary and CV effects of thetest articles. Intravenous MCh is administered at a dose sufficient tocause 80% of the maximal pulmonary constriction in an untreated animal(determined by experimentation in a pilot study using 4 guinea pigs).The pulmonary and CV responses to the MCh determine the potency, safetyand pharmacodynamic effects of test articles.

Guinea Pig Bronchoprotection Protocol-MCh Dose Response:

Test compounds and control (water) were administered to male DunkinHartley guinea pigs (250-350 g) via inhalation. Inhalation dosing wasdone by placing the guinea pigs in a dosing chamber and exposing themfor 25 min to nebulized drug solutions using a Pari nebulizer. Theanimals were then returned to their cages. The chamber wasdecontaminated between uses by washing with water.

Twenty-four hours after dosing and thirty minutes prior to the start ofpulmonary evaluation, the animals were anesthetized with intramuscularketamine (55.8 mg/kg), xylazine (3.9 mg/kg) and acepromazine (1 mg/kg).The trachea was then dissected free and cannulated with a 14 G steeltube connected to a pressure transducer (for the measurement ofpulmonary inflation pressure) and to a constant volume rodent respiratorset to deliver an appropriate tidal volume (2.5 ml) and at a ratedetermined by the animal's weight (100 breath/min). The carotid arterywas cannulated with a 5 U/ml heparin/saline-filled PE-50 cannula andconnected to a pressure transducer for the measurement of blood pressureand heart rate. The jugular vein was catheterized with a saline filledpolyethylene catheter (PE-10) and used to deliver bolus challenges ofthe bronchoconstrictor methacholine (MCh). Intravenous ascending dosesof MCh (1 to 300 μg/kg) were administered, after the response to theprevious dose returned to baseline. The pulmonary inflation pressure andblood pressure were recorded using a Biopac system with the AcqKnowledgesoftware. The animals were euthanized upon completion of the study bycervical dislocation followed by a thoracotomy.

The results of the bronchoprotection studies are shown in FIG. 2. Table3 shows in vivo potency (24 h post inhalation) and duration ofbronchoprotective effects against methacholine-inducedbronchoconstriction in guinea pig.

TABLE 3 Compound ID Potency (ID₅₀) Duration (1) 50 μg/mL  +++ (2) 5μg/mL +++ (3) 3 μg/mL +++ +++ ≧48 h duration

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein by an identifyingcitation are hereby incorporated herein by reference in their entiretiesfor all purposes, to the same extent as if each individual publication,patent, patent application and published patent application wasspecifically and individually indicated to be incorporated by referencein its entirety, for all purposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainchanges and modifications will be practiced. Unless otherwise apparentfrom the context, any step, element, embodiment, feature or aspect ofthe invention can be used with any other. Therefore, the description andexamples should not be construed as limiting the scope of the invention.

What is claimed is:
 1. A method of treating a subject with a respiratorydisease, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising astereochemically pure salt with a stereochemical purity of at least 80%according to Formula (II):

wherein R₁ is independently phenyl or thienyl, both optionallysubstituted with an alkyl, alkoxy, halo, or COOR group; R₂ isindependently phenyl, thienyl, cyclopentyl, cyclohexyl,1-alkylcyclopentyl, 1-alkylcyclohexyl, 1-hydroxycyclopentyl or1-hydroxycyclohexyl, which are optionally substituted with an alkyl,alkoxy, halo, or COOR group; or wherein R₁ and R₂ together are9-xanthenyl, which is substituted on either or both benzene rings withan alkyl, alkoxy, halo, or COOR group; wherein R₃ is OH; wherein R₄ andR₅ are independently lower alkyl, alkoxycarbonylalkyl, aralkyl, oraryloxyalkyl, which are optionally substituted with an alkyl, alkoxy,halo, or COOR group; or R₄ and R₅ together with the ring to which theyare attached form a five- or six-membered ring optionally substitutedwith aryl or aryloxy; wherein R is a lower alkyl; wherein *, **, and ***are each independently a stereocenter, and wherein the stereocenters *,**, and *** are present in one of the following combinations: (i) * is(R), ** is (R), *** is (S), or (ii) * is (S), ** is (S), *** is (R), or(iii) * is (R), ** is (S), *** is (R), or (iv) * is (S), ** is (R), ***is (S); and X⁻ represents a pharmaceutically acceptable anion.
 2. Themethod of claim 1, wherein R₁ is phenyl, optionally substituted with analkyl, alkoxy, halo, or COOR group.
 3. The method of claim 2, wherein R₁is unsubstituted phenyl.
 4. The method of claim 1, wherein R₂ iscyclopentyl.
 5. The method of claim 1, wherein R₄ and R₅ areindependently C₁-C₄ alkyl.
 6. The method of claim 5, wherein both R₄ andR₅ are methyl.
 7. The method of claim 1, wherein R₁ is unsubstitutedphenyl and R₂ cyclopentyl.
 8. The method of claim 1, wherein R₁ isunsubstituted phenyl, R₂ is cyclopentyl, and R₄ and R₅ are independentlyC₁-C₄ alkyl.
 9. The method of claim 1, wherein X⁻ is selected from thegroup consisting of acetate, besylate (benzenesulfonate), benzoate,bicarbonate, bitartrate, bromide, calcium edentate, camphorsulfonate(camsylate), carbonate, chloride, chlorotheophyllinate, citrate,edetate, ethanedisulfonate (edisylate), ethanesulfonate (esylate),fumarate, gluceptate (glucoheptonate), gluconate, glucuronate,glutamate, hexylresorcinate, hydroxynaphthoate, hippurate, iodide,isethionate, lactate, lactobionate, lauryl sulfate (cstolatc), malatc,maleate, mandelate, mesylate, methanesulfonate, methylnitrate,methylsulfate, mucate, naphthoate, napsylate, nitrate, octadecanoate,oleate, oxalate, pamoate, pantothenate, phosphate, polygalacturonate,salicylate, stearate, succinate, sulfate, sulfosalicylate, tannate,tartrate, teoclate, toluenesulfonate (tosylate), and trifluoroacetate.10. The method of claim 1, wherein X⁻ is selected from the groupconsisting of chloride, bromide, iodide, sulfate, methanesulfonate,benzenesulfonate, and toluenesulfonate.
 11. The method of claim 10,wherein X⁻ is bromide.
 12. The method of claim 1, wherein the salt isisolated and has an IC₅₀ for binding to a muscarinic acetylcholinereceptor of about 3 nanomolar or less.
 13. The method of claim 1,wherein the salt is selected from the group consisting of


14. The method of claim 1, wherein the pharmaceutical compositionfurther comprises one or more additional therapeutic agents.
 15. Themethod of claim 1, wherein at least one of the one or more additionaltherapeutic agents is selected from the group consisting of ananti-inflammatory agent, a bronchodilator, an antihistamine, and anantitussive agent.
 16. The method of claim 1, wherein the respiratorydisease is selected from the group consisting of respiratory tractdisorders, chronic obstructive pulmonary disorder (COPD), chronicbronchitis, asthma, chronic respiratory obstruction, pulmonary fibrosis,pulmonary emphysema, rhinorrhea, allergic rhinitis, occupational lungdiseases, pneumoconiosis, black lung disease, silicosis, asbestosis,acute lung injury (ALI), and acute respiratory distress syndrome (ARDS).17. The method of claim 16, wherein the respiratory disease is COPD. 18.The method of claim 1, wherein the pharmaceutical composition isadministered once a day.
 19. The method of claim 1, wherein thepharmaceutical composition is administered by inhalation.
 20. The methodof claim 16, wherein the respiratory disease is asthma.